CN111099575A - Lysosome targeted polarity and viscosity dual-response fluorescent carbon dot, preparation method and application - Google Patents
Lysosome targeted polarity and viscosity dual-response fluorescent carbon dot, preparation method and application Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention provides a lysosome targeted polar and viscosity dual-response fluorescent carbon dot, a preparation method and application thereof, wherein 5-amino-2- (hydroxymethyl) phenylboronic acid cyclic monoester and tartaric acid are mixed according to a molar ratio of 1: dissolving the solution 1 in ultrapure water, transferring the solution into an inner lining of polytetrafluoroethylene, reacting for 12h at 180 ℃ in a vacuum drying oven, cooling, centrifuging to remove larger particles, ultrafiltering to remove smaller raw materials, and freeze-drying to obtain carbon dots. The carbon dots can internally target lysosomes, do not need any target ligand, are not sensitive to pH, and respond to polarity and viscosity; the invention can distinguish normal cells from cancer cells through polarity and viscosity dual responses.
Description
Technical Field
The invention relates to the technical field of fluorescent carbon dots and biosensing, in particular to a lysosome-targeted polarity and viscosity dual-response fluorescent carbon dot, a preparation method and application thereof.
Background
Lysosomes, which are the most acidic and viscous digestive organs in cells, have a variety of functions such as digestion, signaling, plasma membrane repair, intracellular trafficking, homeostasis, and autophagy, and abnormalities thereof may also cause a variety of diseases. Therefore, it is of great interest to develop methods for tracking and visualizing lysosomes.
Cancer is a disease with a high mortality rate, and most of the research on cancer focuses on early diagnosis of cancer. Among them, the main difficulty in cancer diagnosis and treatment is the rapid, sensitive differentiation of cancer cells from normal cells. To date, a variety of clinical detection methods have been developed, such as computed tomography, X-ray, magnetic resonance imaging, ultrasound, atomic force microscopy, modulated raman spectroscopy, and biomarkers, among others. However, these suffer from low signal to noise ratio, radiation overdose, limited design of the designated molecule, cumbersome synthetic procedures, complicated purification procedures, high cost and overexpression. Therefore, there is an urgent need to develop new tools for distinguishing cancer cells from normal cells.
In recent years, fluorescence imaging technology using carbon dots has become a powerful tool for studying biomolecules, living cell processes, and cancer diagnosis due to its excellent selectivity, sensitivity, and spatiotemporal resolution. Although some reported carbon spots can be used to distinguish cancer cells from normal cells, there are no fluorescent carbon spots that have both lysosomal targeting and cancer diagnosis.
Disclosure of Invention
The invention provides a lysosome targeted polarity and viscosity dual-response fluorescent carbon dot, a preparation method and application thereof, and normal cells and cancer cells are distinguished by utilizing the characteristics of low polarity and high viscosity in lysosomes, so that cancer diagnosis is realized.
The technical scheme for realizing the invention is as follows:
a preparation method of lysosome targeted polar and viscosity dual-response fluorescent carbon dots comprises the following steps of mixing 5-amino-2- (hydroxymethyl) phenylboronic acid cyclic monoester with tartaric acid in a molar ratio of 1: dissolving the solution 1 in ultrapure water, transferring the solution into an inner lining of polytetrafluoroethylene, reacting for 12h at 180 ℃ in a vacuum drying oven, cooling, centrifuging to remove larger particles, ultrafiltering to remove smaller raw materials, and freeze-drying to obtain carbon dots.
The synthesis route of the fluorescent carbon dots based on cancer diagnosis is as follows:
the fluorescent carbon dots distinguish normal cells from cancer cells by a dual response of polarity and viscosity in lysosomes. With decreasing polarity (polarity value from 0.3200 to 0.0205) or increasing viscosity (viscosity value from 0.8930 to 856.0), fluorescence at 530nm increased rapidly and exhibited good linearity.
The application specifically comprises:
the change of fluorescence spectra of the carbon dots in solutions with different polarities or solutions with different viscosities is respectively tested, the excitation wavelength of fluorescence is 445 nm, the emission wavelength is 530nm, the fluorescence at 530nm is rapidly increased along with the reduction of the polarities or the increase of the viscosities, and good response is achieved. And simultaneously testing the brightness difference of the carbon dots in different cancer cells and normal cells to achieve the purpose of distinguishing the cancer cells from the normal cells. Has good prospect in biomedical research and diagnosis of related diseases.
The invention has the beneficial effects that: (1) the carbon point synthesis is very simple and convenient to operate; (2) the carbon dots can internally target lysosomes, do not need any target ligand, are not sensitive to pH, and respond to polarity and viscosity; (3) the invention can distinguish normal cells from cancer cells through polarity and viscosity dual responses.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a TEM spectrum of a carbon dot of example 1.
Figure 2 is an XRD pattern of the carbon dots of example 1.
FIG. 3 is an FTIR spectrum of the carbon dots of example 1.
FIG. 4 is an XPS spectrum of carbon dots of example 1.
Fig. 5 is a graph of uv and fluorescence spectra of example 1 carbon dots (50 μ g/mL) in PBS (10mM pH = 7.4).
FIG. 6 is a fluorescence spectrum of pH versus carbon spot of example 1.
FIG. 7 is a fluorescence spectrum of changes with time of fluorescence intensity at 530nm after reaction at 50 μ g/mL carbon spots of amino acids different from 200 μ M, ROS different from 200 μ M, and 1mM cations, respectively, in a PBS buffered (10mM, pH = 7.4) system.
FIG. 8 is a graph of fluorescence spectra and linearity of solutions of different polarity values versus carbon dots of example 1.
FIG. 9 is a graph of the fluorescence spectra and linearity of solutions of different viscosities versus carbon dots of example 1.
FIG. 10 is a graph of the co-localization of the carbon dots of example 1 in HeLa cells with commercial lysosomal dyes.
FIG. 11 is a graph comparing the carbon spots in cancer cells and normal cells in example 1. (a-e) is a cancer cell; (f-h) is a normal cell; (i) is a fluorescence intensity contrast plot.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
The carbon dots are synthesized by the following steps:
adding 0.01 g of 5-amino-2- (hydroxymethyl) phenylboronic acid cyclic monoester and 0.01 g of tartaric acid into a 20 mL weighing bottle, adding 10 mL of ultrapure water, uniformly mixing, putting into an inner lining of polytetrafluoroethylene, reacting for 12h at 180 ℃ in a vacuum drying oven, cooling to room temperature, centrifuging to remove large unreacted particles, ultrafiltering to remove small unreacted raw materials, and freeze-drying to obtain carbon dots.
FIG. 1 is a TEM image of carbon dots, approximately spherical structure, with a statistical size of 7.32 nm, illustrating that carbon dots have been formed.
Fig. 2 is an XRD pattern of carbon dots, showing a broad (002) diffraction peak near 21.3 deg., corresponding to the bragg reflection plane of bragg carbon, and revealing the presence of an amorphous structure.
FIG. 3 is an FTIR spectrum of carbon dots, 3426, 3222, 1743, 1620, 1438, 1295 and 1128 cm-1The results respectively correspond to-OH stretching vibration, N-H stretching vibration, -COOH stretching vibration, N-C = O/C = C stretching vibration, B-O stretching vibration, -OH deforming vibration and C-N/C-O/C-B stretching vibration, and indicate that the two raw materials are combined together to generate carbon dots.
FIG. 4 is an XPS spectrum of carbon dots with atomic percentages of B1s (192.6 eV), C1s (285.0 eV), N1s (402.0 eV), O1s (532.5 eV) being 6.67%, 65.02%, 2.77%, and 25.53%, respectively, further illustrating the generation of carbon dots.
Fig. 5 is a graph of the uv and fluorescence spectra of carbon dots (50 μ g/mL) in PBS (10mM pH = 7.4) with maximum absorption and emission at 490 nm and 530nm, respectively.
Fig. 6 is a fluorescence spectrum of carbon dots at 530nm in PBS buffer solution of pH = 4-8, showing that pH has substantially no effect on carbon dots.
FIG. 7 is a reaction of amino acids with carbon spots of 50 μ g/mL different from 200 μ M, ROS with different from 200 μ M, 1mM cation. To 2 mL of PBS buffered (10mM, pH = 7.4) system containing 50 μ g/mL carbon dots, 20 μ L of 20 mM analyte: 1: carbon points; 2: threonine; 3: arginine; 4: methionine; 5: (ii) proline; 6: glycine; 7: lysine; 8: leucine; 9: tryptophan; 10: isoleucine; 11: alanine; 12: (ii) histidine; 13: glutamic acid; 14: aspartic acid; 15: valine; 16: serine; 17: (ii) cysteine; 18: homocysteine; 19: glutathione; 20: o is2·-；21：H2O2；22：NaNO2；23：·OH；24：NaClO；25：KCl；26：CaCl2；27：NaCl；28：MgCl2；29：AlCl3；30：ZnSO4；31：FeCl3；32：FeCl2；33：CuSO4；34：NiCl2；35：MnCl2Fluorescence spectrometry was performed. The fluorescence intensities at 530nm for the different analytes were also compared. Experimental data indicate that the carbon dots do not react with these analytes.
Example 2
FIG. 8 is a graph (a) showing the fluorescence spectrum and linear relationship between solutions of different polarity values and the carbon dots of example 1. Respectively adding 2 mL of 1, 4-dioxane and water in different proportions into the cuvette (according to the condition that the content of the 1, 4-dioxane is from 0% to 99%, the corresponding polarity value is reduced from 0.3200 to 0.0205), adding 10 mu L of carbon dot storage solution with the concentration of 10 mg/mL, and carrying out fluorescence spectrum test after uniformly mixing. The experimental result shows that the fluorescence at 530nm is gradually increased along with the reduction of the polarity value of the solvent, and the linear relation is R2=0.9910, indicating that the carbon dot responds well to polarity.
FIG. 9 is a graph of the fluorescence spectrum (a) and the linear relationship (b) of solutions of different viscosities to the carbon dots of example 1. Adding 2 mL of glycerol and water in different proportions into the cuvette respectively (the content of the glycerol is increased from 0% to 99% corresponding to a viscosity value of 0.8930 to 856.0), adding 10 mu L of carbon dot storage solution with the concentration of 10 mg/mL, and carrying out fluorescence spectrum test after uniformly mixing. The experimental result shows that the fluorescence at 530nm is gradually increased along with the increase of the viscosity, and the linear relation is R2= 0.9633, indicating that the carbon point responds well to viscosity.
Example 3
FIG. 10 is a graph of the co-localization of the carbon dots of example 1 in HeLa cells with commercial lysosomal dyes. HeLa cells were incubated with 50 μ g/mL carbon spots for 20 min with a commercial lysosomal dye LysoTrackerTMDeep Red (50 nM), incubation for 15 min, PBS washing three times, and good overlap of carbon spots with commercial lysosomal probes was observed in confocal microscopy, indicating that carbon spots can target lysosomes.
FIG. 11 is a graph comparing the carbon spots in cancer cells and normal cells in example 1. (a-e) is a cancer cell; (f-h) is a normal cell; (i) is a fluorescence intensity contrast plot. The HeLa cells are incubated for 20 min by using 50 mug/mL carbon dots, PBS is washed for three times, and confocal imaging finds that the brightness of the carbon dots in the cancer cells is far higher than that of normal cells, which indicates that the carbon dots can distinguish the normal cells from the cancer cells.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A preparation method of lysosome targeted polarity and viscosity dual-response fluorescent carbon dots is characterized by comprising the following steps: dissolving 5-amino-2- (hydroxymethyl) phenylboronic acid cyclic monoester and tartaric acid in ultrapure water, reacting the mixed solution, cooling, centrifuging, ultrafiltering, and freeze-drying to obtain the carbon dots.
2. The method for preparing a lysosome-targeted polar and viscosity dual-response fluorescent carbon dot according to claim 1, characterized in that: the molar ratio of the 5-amino-2- (hydroxymethyl) phenyl boronic acid cyclic monoester to the tartaric acid is 1: 1.
3. the method for preparing a lysosome-targeted polar and viscosity dual-response fluorescent carbon dot according to claim 1, characterized in that: and transferring the mixed solution into polytetrafluoroethylene, and reacting for 12 hours in a vacuum drying oven at 180 ℃.
4. A fluorescent carbon dot prepared according to any one of claims 1 to 3.
5. Use of the fluorescent carbon dot of claim 4 for targeted detection of polarity and viscosity in lysosomes.
6. Use according to claim 5, characterized in that: respectively adding 2 mL of 1, 4-dioxane and water in different proportions into a cuvette, adding 10 mu L of carbon dot storage liquid with the concentration of 10 mg/mL, and testing a fluorescence spectrum at 530nm after uniformly mixing.
7. Use according to claim 6, characterized in that: when the polarity of the solution is reduced from 0.3200 to 0.0205, the fluorescence at 530nm increases rapidly.
8. Use according to claim 5, characterized in that: adding 2 mL of glycerol and water in different proportions into the cuvette respectively, adding 10 muL of carbon dot storage solution with the concentration of 10 mg/mL, and testing a fluorescence spectrum at 530nm after uniformly mixing.
9. Use according to claim 8, characterized in that: the solution viscosity increased from 0.8930 to 856.0, with a rapid increase in fluorescence at 530 nm.
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